Beverage manufacture and cold aseptic bottling using peroxyacid antimicrobial composition

ABSTRACT

A peroxyacid antimicrobial concentrate and use composition is provided comprising a C 1  to C 4  peroxycarboxylic acid or a C 1  to C 4  peroxycarboxylic acid combined with a C 6  to C 18  peroxyacid in beverage processing. The combination of these materials produces a synergistic effect, providing a much more potent biocide than can be obtained by using these components separately. Other components can be added to the composition such as hydrotrope coupling agents, stabilizers, etc. An effective antimicrobial use solution is formed at low concentrations when the concentrate composition is diluted with water to a pH in the range of about 2 to 8. Sanitizing of substantially fixed, “in-place” processing lines in dairies, breweries, and other food and beverage processing operations is one utility of the composition. Another utility is in processes including aseptic cold filling of beverage containers such as cans, glass bottles or two liter PET bottles.

FIELD OF THE INVENTION

The invention relates generally to processes using antimicrobial orbiocidal compositions. More particularly, the invention relates toperoxyacid antimicrobial concentrates and use solutions which cansanitize various surfaces such as facilities, containers or equipmentfound in the food or beverage processing in food service industries ator near ambient temperatures.

BACKGROUND OF THE INVENTION

Numerous classes of chemical compounds exhibit varying degrees ofantimicrobial or biocidal activity. Antimicrobial compositions areparticularly needed in the food and beverage industries to clean andsanitize processing facilities such as pipelines, tanks, mixers, etc.and continuously operating homogenation or pasteurization apparatus.Sanitizing compositions have been formulated in the past to combatmicrobial growth in such facilities. For example, Grosse-Böwing, U.S.Pat. Nos. 4,051,058 and 4,051,059 teaches peracetic acid materials.These peroxy-containing compositions are known for use in the productionof microbicidal agents. One such composition is disclosed inGrosse-Böwing et al. contains peracetic acid, acetic acid or mixtures ofperacetic and acetic acid, hydrogen peroxide, anionic surface activecompounds such as sulfonates and sulfates, and water. Wang, U.S. Pat.No. 4,404,040, teaches a short chain fatty acid sanitizing compositioncomprising an aliphatic short chain fatty acid, a hydrotrope solubilizercapable of solubilizing the fatty acid in both the concentrate and usesolution, and a hydrotrope compatible acid so that the use solution hasa pH in the range of 2.0 to 5.0.

Peracetic acid has been shown to be a good biocide, but only at fairlyhigh concentrations (generally greater than 100 part per million (ppm)).Similarly, peroxyfatty acids have also been shown to be biocidal, butonly at high concentrations (greater than 200 ppm), such as in thecomposition disclosed in European Patent Application No. 233,731.Antimicrobial compositions having low use concentrations (less than 100ppm) which effectively kill microbes are particularly desirable. Lowconcentrations minimize use cost, surface corrosion, odor, carryover ofbiocide into foods and potential toxic effects to the user. Therefore, acontinuing need exists to provide such an antimicrobial composition foruse in food processing, food service and health care facilities. Incontrast to the prior art, the composition of the present invention hasthe unique advantage of having antimicrobial or biocidal activity at lowlevel use concentrations.

SUMMARY OF THE INVENTION

The invention is a peroxyacid antimicrobial concentrate and diluted enduse composition comprising an effective microbicidal amount of a C₁-C₄peroxycarboxylic acid or an effective microbicidal amount of a C₁-C₄peroxycarboxylic acid combined with an effective microbicidal amount ofa C₆-C₁₈ peroxyacid. The concentrate composition can be diluted with amajor proportion of water to form an antimicrobial sanitizing usesolution having a pH in the range of about 2 to 8, with a C₁-C₄peroxycarboxylic acid concentration of at least about 10 ppm, preferablyabout 10 to 75 ppm, and a C₆-C₁₈ peroxyacid concentration of at leastabout 1 ppm, preferably about 1 to 25 ppm. Other components may be addedsuch as a hydrotrope coupling agent for solubilizing the peroxyfattyacid in the concentrate form and when the concentrate composition isdiluted with water. In contrast to the prior art, we have discoveredthat at a low pH, (e.g. preferably less than 5) C₆-C₁₈ peroxyacids suchas peroxyfatty acids are very potent biocides at low levels. When usedin combination with a C₁-C₄ peroxycarboxylic acid such as peroxyaceticacid, a synergistic effect is obtained, providing a much more potentbiocide than can be obtained by using these components separately. Thismeans that substantially lower concentrations of biocide can be used toobtain equal biocidal effects, leading to lower costs of the product andless potential for corrosion. As the term is used herein, a C₆-C₁₈peroxyacid (or peracid) is intended to mean the product of the oxidationof a C₆-C₁₈ acid such as a fatty acid, or a mixture of acids, to form aperoxyacid having from about 6 to 18 carbon atoms per molecule. TheC₁-C₄ peroxycarboxylic acid is intended to mean the product of oxidationof a C₁-C₄ carboxylic acid, or a mixture thereof. This includes bothsimple and substituted C₁-C₄ carboxylic acids.

A method of sanitizing facilities or equipment comprises the steps ofcontacting the facilities or equipment with the use solution made fromthe above concentrate composition of the invention at a temperature inthe range of about 0° C. to 80° C., preferably 20° C. to 40° C. andoften at ambient or room temperature conditions. The composition is thencirculated or left in contact with the container, facilities orequipment for a time sufficient to sanitize (generally at least 30seconds) and the composition is thereafter drained or removed from thecontainer, facilities or equipment. We have found that the methods ofthe invention are capable of killing a variety of microorganismsincluding bacteria, yeast and mold. In particular the methods aresurprisingly effective for aseptic cold filling of beverage containers.The compositions are effective against either fungal genus, Chaetomiumor Arthrinium, that are a problem in bottling operations.

One aspect of the invention is the novel, antimicrobial concentratecomposition which is capable of being diluted with a major proportion ofwater to form a sanitizing use solution. A further aspect of theinvention is an aqueous antimicrobial sanitizing use solution which isparticularly suited for “in-place” cleaning applications. A furtheraspect of the invention is a method of employing the use solution of theinvention in the cleaning or sanitizing of various process facilities orequipment as well as other surfaces.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE is a diagram of a beverage plant, including a cold asepticfilling plant, in which either carbonated or non-carbonated beveragescan be prepared and bottled.

DETAILED DESCRIPTION OF THE INVENTION

The invention resides in a peroxyacid antimicrobial concentrate and usecomposition comprising an effective microbicidal amount of a C₁-C₄peroxycarboxylic acid or an effective microbicidal amount of a C₁-C₄peroxycarboxylic acid combined with an effective microbicidal amount ofa C₆-C₁₈ peroxyacid. We have found that combining these acids produces asynergistic effect, producing a much more potent biocide than can beobtained by using these components separately. The concentratecomposition can be diluted with a major proportion of water to form anantimicrobial sanitizing use solution having a pH in the range of about2 to 8. The sanitizing use solution can be used effectively to clean orsanitize facilities and equipment used in the food processing, foodservice and health care industries.

Peroxyacids

Peroxy-containing concentrates which are stable in storage can beobtained which are useful for the production and supplementation ofmicrobicides based in aliphatic monopercarboxylic acids. Theseperoxy-containing concentrates are characterized by a content of 0.5% to20% by weight of a per acid with 2 to 3 carbon atoms and/or thecorresponding aliphatic noncarboxylic acid, as well as 25% to 40% byweight of H₂O₂, and the balance, water.

More particularly, the invention relates to a peroxy-containingconcentrate, stable in storage, comprising from 0.5% to 20% by weight ofan acid selected from the group consisting of peracetic acid, aceticacid, perpropionic acid, propionic acid, mixtures of peracetic acid andacetic acid, and mixtures of perpropionic acid and propionic acid, from25% to 40% by weight of H₂O₂, from 0 to 5% by weight of an anionicsurface-active compound selected from the group consisting of sulfonatesand sulfates, and the remainder to 100% by weight, water. Preferably,the storage-stable peroxy-containing concentrates contain from 5% to 10%by weight of component (1), and a molar excess of H₂O₂ with reference tothe acid component (1), calculated as the monocarboxylic acid, in amolar ratio of at least 2:1, preferably 3:1 to 50:1. When the anionicsurface-active compound of the sulfonate and the sulfate type ispresent, it is preferably in an amount of from 0.5% to 5% by weight.

The production is effected in a simple manner by mixing an H₂O₂solution, preferably with a concentration of about 33% by the weightwith per acid such as peracetic acid and, optionally, acetic acid. Themixtures can also be produced in an advantageous manner by adding thecorresponding amount of the acid, such as acetic acid, to theconcentrated H₂O₂ solution. Since the products mainly are not used atonce, but are first stored, a corresponding content of peracetic acid isformed when acetic acid is employed. The formation of peracetic acid canbe catalytically accelerated, if desired, by adding a small amount of amineral acid (0.1% to 1% by weight). In general, however, such anaddition is not necessary for the above-mentioned reasons.

Such concentrates, which are produced, for example, from 30% of H₂O₂, 5%of acetic acid and 65% of water, have no annoying odor and are easy tohandle, that is, they can be easily diluted to the concentration of 0.1%to 1%, as they are used in food technology and in the medical field,without requiring special precautions.

In view of the many possible uses of the above-describedperoxy-containing concentrates as functional agents, for example, forthe oxidation of organic material in general, or for the treatment ofhair, straw and textiles, as well as the preparation of microbicides andvirucides, it is sometimes of advantage to add a wetting agent in orderto improve the desired properties further.

It was found that stable concentrates of the above-described type can beobtained if anionic surface-active compounds of the sulfonate andsulfate type, such as alkylbenzene sulfonates having 6 to 18 carbonatoms in the alkyl, alkyl sulfates and/or alkane sulfonates (each having8 to 22 carbon atoms in the alkyl or alkane groups) are added in amountsof 0.05% to 5% by weight.

The alkylbenzene sulfonates which can be employed are preferably thosewhich contain an alkyl radical of 6 to 18 carbon atoms, preferably 9 to15 carbon atoms. Instead of the alkylbenzene sulfonates, alkyl sulfatesor alkane sulfonates with an alkyl or alkane radical of the chain length12 to 18 carbon atoms, can be employed. If desired, mixtures of theabove-mentioned anionic surface-active compounds can naturally also beused. It was found that, with the above-mentioned additives, theconcentrates remain stable over long periods of time and that thecontent of peracetic acid in the concentrate thus also remains constant.However, if soaps or the conventional nonionic surface-active compoundsare employed as the surface-active additive, a sufficient stability isnot achieved.

The new stable peroxy-containing concentrates are useful in theproduction of functional agents which can be used for all purposes wherean oxidizing effect is to be achieved and the disadvantages of the knownpure per acids render their application difficult or impossible. Theconcentrates have, furthermore, the advantage that they can be employedto produce function agents for static disinfections to prevent thegrowth of germs on machines after cleaning, particularly in the foodindustry. Due to their content of H₂O₂, they have a long-term effect onmost microorganisms. The pH-value of the solution produced is stillweakly acid, and the residues of acetic acid after the disinfection areextremely small, so that the agents are also suitable for disinfectionswhere rinsing is no longer necessary.

The peroxyacid sanitizing materials of the invention can be used in themanufacture of beverage materials including fruit juice, dairy products,malt beverages, bottled water products, teas and soft drinks. Thematerials can be used to sanitize bottles, pumps, lines, tanks andmixing equipment used in the manufacture of such beverages. Further, theperoxyacid materials can be used in aseptic, cold filling operations inwhich the interior of the beverage container is sanitized prior tofilling. In such operations, a beverage container is contacted with thesanitizing peroxyacid material, typically using a spray device tointimately contact the inside of then container with the peroxyacid, forsufficient period of time to reduce microorganism populations within thecontainer. The container is then emptied of the amount of sanitizerused. After emptying, the container can then be commonly rinsed withpotable water or sterilized water and again emptied. After rinsing, thecontainer is then filled with the liquid beverage. The container is thenscaled, capped or closed and then packed for shipment for ultimate sale.The sealed container can be autoclaved or retorted for addedmicroorganism kill.

In beverage manufacture, we have found that a fungal microorganisms ofthe genus Chaetomium or Arthriniurm can be a significant problem inbottling processes, particularly in cold aseptic bottling processes. Theperoxyacid sanitizer materials of the invention can be used for thepurpose of controlling or substantially reducing (by more than a 5 log₁₀reduction) the number of Chaetomium or Arthrinium microorganisms inbeverage bottling lines using cold aseptic bottling techniques. In suchtechniques, metallic, aluminum or steel cans can be filled, glassbottles or containers can be filled or plastic (PET or PBT or PENbottles) can be filled using cold aseptic filling techniques. In suchprocesses, the peroxyacid materials of the invention can be used tosanitize the interior of the beverage containers prior to filling withthe carbonated beverage. Typical carbonated beverages in thisapplication include cola beverage, fruit beverages, ginger alebeverages, root beer beverages, iced tea beverages which may benon-carbonated, and other common beverages considered soft drinks. Theperoxyacid materials of the invention can be used to sanitize both thetanks, lines, pumps, and other equipment used for the manufacture andstorage of the soft drink material and also used in the bottling orcontainers for the beverages. The peroxyacid sanitizing materials areuseful for killing both bacterial and fungal microorganisms that can bepresent on the surfaces of the production equipment and beveragecontainers.

The present invention is based upon the surprising discovery that when aC₅-C₁₈ peroxyacid is combined with a C₁-C₄ peroxycarboxylic acid, asynergistic effect is produced and greatly enhanced antimicrobialactivity is exhibited when compared to the C₅-C₁₈ peroxyacid or theC₁-C₄ peroxycarboxylic acid alone. The present blend of a C₅-C₁₈peroxyacid and a C₁-C₄ peroxycarboxylic acid can effectively killmicroorganisms (e.g., a 5 log₁₀ reduction in 30 seconds) from aconcentration level below 100 ppm and as low as 20 ppm of the peroxyacidblend.

A variety of a C₅-C₁₈ or a C₆-C₁₈ peroxyacids may be employed in thecomposition of the invention such as peroxyfatty acids, monoperoxy ordiperoxydicarboxylic acids, and peroxyaromatic acids. The C₆-C₁₈peroxyacids employed in the present invention may be structurallyrepresented as follows: R₁—CO₃H, wherein R₁ is a hydrocarbon moietyhaving from about 5 to 17 carbon atoms (a C₈ peroxyacid is generallyrepresented structurally as C₇—CO₃H ). R₁ may have substituents in thechain, e.g., —OH, CO₂H, or heteroatoms (e.g., —0—as in alkylethercarboxylic acids), as long as the antimicrobial properties of theoverall composition are not significantly affected. It should berecognized that “R₁” substituents or heteroatoms may change the overallacidity (i.e., pKa) of the carboxylic acids herein described. Suchmodification is within the contemplation of the present inventionprovided the advantageous antimicrobial performance is maintained.Furthermore, R₁ may be linear, branched, cyclic or aromatic. Preferredhydrocarbon moieties (i.e. preferred R₁'s) include linear, saturated,hydrocarbon aliphatic moieties having from 7 to 11 carbon atoms (or 8 to12 carbon atoms per molecule).

Specific examples of suitable C₆-C₁₈ carboxylic acids which can bereacted with hydrogen peroxide to form peroxyacids include suchsaturated acids as hexanoic (C₆), enanthic (heptanoic) (C₇), caprylic(octanoic) (C₈), pelargonic (nonanoic) (C_(g)), capric (decanoic) (C₁₀),undecyclic (undecanoic) (C₁₁), lauric (dodecanoic) (C₁₂), trideclic(tridecanoic) (C₁₃), myristic (tetradecanoic) (C₁₄), palmitic(hexadecanoic) (C₁₆), and stearic (octodecanoic) (C₁₈). These acids canbe derived from both natural and synthetic sources. Natural sourcesinclude animal and vegetable fats or oils which should be fullyhydrogenated. Synthetic acids can be produced by the oxidation ofpetroleum wax. Particularly preferred peroxyfatty acids for use in thecomposition of the invention are linear monoperoxy aliphatic fatty acidssuch as peroxyoctanoic acid, peroxydecanoic acid, or mixtures thereof.

Other suitable C₆-C₁₈ peroxyacids are derived from the oxidation ofdicarboxylic acids and aromatic acids. Suitable dicarboxylic acidsinclude adipic acid (C₆), glutaric acid (C₅) and sebacic acid (C₁₀). Anexample of a suitable aromatic acid is benzoic acid. These acids can bereacted with hydrogen peroxide to form the peroxyacid form suitable foruse in the composition of the invention. Preferred peroxyacids in thisgroup include monoperoxy- or diperoxyadipic acid, monoperoxy- ordiperoxysebacic acid, and peroxybenzoic acid.

The above peroxyacids provide antibacterial activity against a widevariety of microorganisms, such as gram positive (e.g., Staphylococcusaureus) and gram negative (e.g., Escherichia coli) microorganisms,yeast, molds (e.g. Chaetomium, Arthrinium and similar genera), bacterialspores, etc. When the above C₆-C₁₈ peroxyacids are combined with a C₁-C₄peroxycarboxylic acid, greatly enhanced activity is shown compared tothe C₁-C₄ peroxycarboxylic acid alone or the C₆-C₁₈ peroxyacid alone.

The C₁-C₄ peroxycarboxylic acid component can be derived from a C₁-C₄carboxylic acid or dicarboxylic acid by reacting the acid with hydrogenperoxide. Examples of suitable C₁-C₄ carboxylic acids include aceticacid, propionic acid, glycolic acid, and succinic acid. Preferable C₁-C₄peroxycarboxylic acids for use in the composition of the inventioninclude peroxyacetic acid, peroxypropionic acid, peroxyglycolic acid,peroxysuccinic acid, or mixtures thereof. The antimicrobial concentrateof the present invention can comprise about 0.01 to 10 wt-%, preferablyabout 0.05 to 5 wt-%, and most preferably about 0.1 to 2 wt-% of aC₆-C₁₈ peroxyacid, and about 0.1 to 25 wt-%, preferably about 0.5 to 20wt-%, and most preferably about 1 to 15 wt-% of a C₁-C₄ peroxycarboxylicacid. The concentrate composition preferably has a weight ratio of C₁-C₄peroxycarboxylic acid to C₆-C₁₈ peroxyacid of about 15:1 to 3:1. Theconcentrate contains sufficient acid so that the end use solution has apH of about 2 to 8, preferably about 3 to 7. Some acidity may come froman inert acidulant which may be optionally added (e.g, phosphoric acid).

The peroxyacid components used in the composition of the invention canbe produced in a simple manner by mixing a hydrogen peroxide (H₂O₂)solution with the desired amount of acid. With the higher molecularweight acids, a hydrotrope coupler may be required to help solubilizethe acid. The H₂O₂ solution also can be added to previously madeperoxyacids such as peracetic acid or various peroxyacids to produce theperoxyacid composition of the invention. The concentrate can containabout 1 to 50 wt-%, preferably about 5 to 25 wt-% of hydrogen peroxide.

The concentrate composition can further comprise a free C₆-C₁₈carboxylic acid, a free C₁-C₄ carboxylic acid, or mixtures thereof. Thefree acids will preferably correspond to the starting materials used inthe preparation of the peroxyacid components. The free C₆-C₁₈ carboxylicacid is preferably linear and saturated, has 8 to 12 carbon atoms permolecule, and can also comprise a mixture of acids. The free C₆-C₁₈carboxylic acid and free C₁-C₄ carboxylic acid can be present as aresult of an equilibrium reaction with the hydrogen peroxide to form theperoxyacids.

Optional Components

Various optional materials may be added to the composition of theinvention to help solubilize the fatty acids, restrict or enhance theformation of foam, to control hard water, to stabilize the composition,or to further enhance the antimicrobial activity of the composition. Thecomposition of the invention can contain a surfactant hydrotropecoupling agent or solubilizer that permits blending short chain peroxyacids in aqueous liquids. Functionally speaking, the suitable couplerswhich can be employed are non-toxic and retain the fatty acid and theperoxy acid in aqueous solution throughout the temperature range andconcentration to which a concentrate or any use solution is exposed.

Any hydrotrope coupler may be used provided it does not react with theother components of the composition or negatively affect theantimicrobial properties of the composition. Representative classes ofhydrotropic coupling agents or solubilizers which can be employedinclude anionic surfactants such as alkyl sulfates and alkanesulfonates, linear alkyl benzene or naphthalene sulfonates, secondaryalkane sulfonates, alkyl ether sulfates or sulfonates alkyl phosphatesor phosphonates, dialkyl sulfosuccinic acid esters, sugar esters (e.g.,sorbitan esters) and C₈-C₁₀ alkyl glucosides. Preferred coupling agentsfor use in the present invention include noctanesulfonate, available asNAS 8D from Ecolab, and the commonly available aromatic sulfonates suchas the alkyl benzene sulfonates (e.g. xylene sulfonates) or naphthalenesulfonates.

Some of the above hydrotropic coupling agents independently exhibitantimicrobial activity at low pH. This adds to the efficacy of thepresent invention, but is not the primary criterion used in selecting anappropriate coupling agent. Since it is the presence of peroxy acid inthe protonated neutral state which provides biocidal activity, thecoupling agent should be selected not for its independent antimicrobialactivity but for its ability to provide effective interaction betweenthe substantially insoluble peroxy acids described herein and themicroorganisms which the present compositions control.

The hydrotrope coupling agent can comprise about 0.1 to 30 wt-%,preferably about 1 to 20 wt-%, and most preferably about 2 to 15 wt-% ofthe concentrate composition.

Compounds such as mono, di and trialkyl phosphate esters may be added tothe composition to suppress foam. Such phosphate esters would generallybe produced from aliphatic linear alcohols, there being from 8 to 12carbon atoms in the aliphatic portions of the alkyl phosphate esters.Alkyl phosphate esters possess some antimicrobial activity in their ownright under the conditions of the present invention. This antimicrobialactivity also tends to add to the overall antimicrobial activity of thepresent compositions even though the phosphate esters may be added forother reasons. Furthermore, the addition of nonionic surfactants wouldtend to reduce foam formation herein. Such materials tend to enhanceperformance of the other components of the composition, particularly incold or soft water. A particularly useful nonionic surfactant for use asa defoamer is nonylphenol having an average of 12 moles of ethyleneoxide condensed thereon, it being encapped with a hydrophobic portioncomprising an average of 30 moles of propylene oxide.

Chelating agents can be added to the composition of the invention toenhance biological activity, cleaning performance and stability of theperoxyacids. For example, 1-hydroxyethylidene-1,1-diphosphonic acidcommercially available from the Monsanto Company under the designation“DEQUEST” has been found to be effective. Chelating agents can be addedto the present composition to control or sequester hardness ions such ascalcium and magnesium. In this manner both detergency and sanitizationcapability can be enhanced.

Other materials which are sufficiently stable at the low pH contemplatedby the present composition may be added to the composition to impartdesirable qualities depending upon the intended ultimate use. Forexample, phosphoric acid (H₃PO₄) can be added to the composition of theinvention. Additional compounds can be added to the concentrate (andthus ultimately to the use solution) to change its color or odor, toadjust its viscosity, to enhance its thermal (i.e., freeze-thaw)stability or to provide other qualities which tend to make it moremarketable.

The composition of the invention can be made by combining by simplemixing an effective amount of a C₅-C₁₈ or C₆-C₁₈ peroxyacid such as aperoxyacid with some source of a C₁-C₄ peroxycarboxylic acid such asperoxyacetic acid. This composition would be formulated with preformedperoxyacid and preformed peroxyacetic acid. A preferred composition ofthe invention can be made by mixing a C₁-C₄ carboxylic acid, a C₆-C₁₈carboxylic acid, a coupler and a stabilizer and reacting this mixturewith hydrogen peroxide. A stable equilibrium mixture is producedcontaining a C₁-C₄ peroxycarboxylic acid and a C₆-C₁₈ peroxyacid byallowing the mixture to stand for from one to seven days at 15° C. to25° C. As with any aqueous reaction of hydrogen peroxide with a freecarboxylic acid, this gives a true equilibrium mixture. In this case,the equilibritun mixture will contain hydrogen peroxide, a C₁-C₄carboxylic acid, a C₆-C₁₈ carboxylic acid, a C₁-C₄ peroxycarboxylicacid, a C₆-C₁₈ peroxyacid, water, and various couplers and stabilizers.

By using the above approach, the composition of the invention can beformulated by merely mixing readily available raw materials, e.g.,acetic acid, hydrogen peroxide and fatty acid. By allowing solution timefor equilibrium to be obtained, the product containing both of theactive biocides is obtained. In varying the ratio of C₁-C₄ carboxylicacid to C₆-C₁₈ carboxylic acid, it is easy to vary the ratio of C₁-C₄peroxycarboxylic acid to C₆-C₁₈ peroxyacid.

Concentrate and Use Compositions

The present invention contemplates a concentrate composition which isdiluted to a use solution prior to its utilization as a sanitizer.Primarily for reasons of economics, the concentrate would normally bemarketed and the end user would dilute the concentrate with water to ause solution. A preferred antimicrobial concentrate compositioncomprises about 0.01 to 10 wt-%, preferably about 0.05 to 5 wt-%, of aC₆-C₁₈ peroxyfatty acid, about 0.1 to 25 wt-%, preferably about 0.5 to20 wt-%, of a C₁-C₄ peroxycarboxylic acid, about 0.1 to 30 wt-% of ahydrotrope coupling agent, and about 1 to 50 wt-% of hydrogen peroxide.Other acidulants may optionally be employed in the composition such asphosphoric acid.

The level of active components in the concentrate composition isdependent upon the intended dilution factor and desired acidity in theuse solution. The C₆-C₁₈ peroxyacid component is generally obtained byreacting a C₆-C₁₈ carboxylic acid with hydrogen peroxide in the presenceof a C₁-C₄ carboxylic acid. The resulting concentrate is diluted withwater to provide the use solution. Generally, a dilution of 1 fluid oz.to 4 gallons (i.e. dilution of 1 to 500 by volume) or to 8 gallons (i.e.dilution of 1 to 1,000 by volume) of water can be obtained with 2% to20% total peroxyacids in the concentrate. Higher use dilution can beemployed if elevated use temperature (greater than 20° C.) or extendedexposure time (greater than 30 seconds) are also employed. In itsintended end use, the concentrate is diluted with a major proportion ofwater and used for purposes of sanitization. The typical concentratecomposition described above is diluted with available tap or servicewater to a formulation of approximately 1 oz. concentrate to 8 gallonsof water. An aqueous antimicrobial sanitizing use solution comprises atleast about 1 part per million (ppm), preferably about 2 to 10 ppm of aC₆-C₁₈ peroxyacid, and at least about 10 ppm, preferably about 20 to 50ppm of a C₁-C₄ peroxycarboxylic acid. The weight ratio of C₆-C₁₈peroxyacid to C₁-C₄ peroxycarboxylic acid ranges from about 0.01 to 0.5parts, preferably about 0.02 to 0.2 parts of C₆-C₁₈ peroxyacid per partof C₁-C₄ peroxycarboxylic acid. Preferably the total peroxyacidconcentration in the use solution is less than about 75 ppm, and mostpreferably between about 5 to 50 ppm. Higher levels of peroxyacids canbe employed in the use solution to obtain disinfecting or sterilizingresults.

The aqueous use solution can further comprise at least about 1 ppm,preferably about 2 to 20 ppm, of a hydrotrope coupling agent, at leastabout 1 ppm, preferably about 2 to 200 ppm of hydrogen peroxide, and atleast about 1 ppm, preferably about 2 to 200 ppm of a free C₆-C₁₈carboxylic acid, a free C₁-C₄ carboxylic acid, or mixtures thereof. Theaqueous use solution has a pH in the range of about 2 to 8, preferablyabout 3 to 7.

Methods of Use

As noted above, the present composition is useful in the cleaning orsanitizing of containers processing facilities or equipment in the foodservice, food processing or health care industries. Examples of processfacilities in which the composition of the invention can be employedinclude a milk line dairy, a continuous brewing system, food processinglines such as pumpable food systems and beverage lines, etc. Foodservice wares can also be disinfected with the composition of theinvention. The composition is also useful in sanitizing or disinfectingsolid surfaces such as floors, counters, furniture, medical tools andequipment, etc., found in the health care industry. Such surfaces oftenbecome contaminated with liquid body spills such as blood, otherhazardous body fluids or mixtures thereof. Containers include glassbottles, PVC or polyolefin film sacks, cans, polyester, PEN or PETbottles of various volumes (100 ml to 2 liter, etc.), one gallon milkcontainers, paper board juice or milk containers, etc.

Generally, the actual cleaning of the in-place system or other surface(i.e., removal of unwanted offal therein) is accomplished with adifferent material such as a formulated detergent which is introducedwith heated water. After this cleaning step, the instant sanitizingcomposition would be applied or introduced into the system at a usesolution concentration in unheated, ambient temperature water. Thepresent sanitizing composition is found to remain in solution in cold(e.g., 40° F./4° C.) water and heated (e.g., 140° F./60° C.) water.Although it is not normally necessary to heat the aqueous use solutionof the present composition, under some circumstances heating may bedesirable to further enhance its antimicrobial activity. These materialsare useful at any conceivable temperatures.

A method of sanitizing substantially fixed in-place process facilitiescomprises the following steps. The use composition of the invention isintroduced into the process facilities at a temperature in the range ofabout 4° C. to 60° C. After introduction of the use solution, thesolution is held in a container or circulated throughout the system fora time sufficient to sanitize the process facilities (i.e., to killundesirable microorganisms). After the surfaces have been sanitized bymeans of the present composition, the use solution is drained. Uponcompletion of the sanitizing step, the system optionally may be rinsedwith other materials such as potable water. The composition ispreferably circulated through the process facilities for 10 minutes orless.

The composition may also be employed by dipping food processingequipment into the use solution, soaking the equipment for a timesufficient to sanitize the equipment, and wiping or draining excesssolution off the equipment, The composition may be further employed byspraying or wiping food processing surfaces with the use solution,keeping the surfaces wet for a time sufficient to sanitize the surfaces,and removing excess solution by wiping, draining vertically, vacuuming,etc.

The composition of the invention may also be used in a method ofsanitizing hard surfaces such as institutional type equipment, utensils,dishes, health care equipment or tools, and other hard surfaces. Thecomposition may also be employed in sanitizing clothing items or fabricwhich have become contaminated. The use composition is contacted withany of the above contaminated surfaces or items at use temperatures inthe range of about 4° C. to 60° C., for a period of time effective tosanitize, disinfect, or sterilize the surface or item. For example, theconcentrate composition can be injected into the wash or rinse water ofa laundry machine and contacted with contaminated fabric for a timesufficient to sanitize the fabric. Excess solution can then be removedby rinsing or centrifuging the fabric.

As the term “sanitizing” is used in the method of the instant invention,it means a reduction in the population numbers of undesirablemicroorganisms by about 5 powers of 10 or greater (i.e., at least 5orders of magnitude) after a 30 second exposure time. It is to beemphasized that the instant use solution provides cleaning as well assanitizing performance although its primary utility is sanitizing. Thecomposition may also be used-to achieve disinfection or sterilization(i.e., elimination of all microorganisms) by employing higher levels ofperoxyacids in the use solution.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that these Examples suggest many other waysin which the present invention could be practiced.

DETAILED DESCRIPTION OF THE DRAWINGS

The Figure shows a schematic for a bottle spraying/bottling operationusing a peroxyacid active sanitizer materials including a cold asepticoperation. In the figure, a plant 100 that can contact beverage bottleswith a peroxyacid sanitizer for a sanitizing regiment is shown. In thefigure, bottles 110 are passed through a sterilizing tunnel 102. Thesanitized bottles 110 a then pass through a rinsing tunnel 103 andemerge as sanitized rinsed bottles 110 b. In the process, bulkperoxyacid sanitizer is provided in a drum or container 104 and added toa holding tank 101 at an effective concentration comprising about 0.1 toabout 5 wt %, preferably 3 wt % to about 4 wt %. Commonly, the materialsmaintained at a temperature of about 22° C. in tank 101. To obtain theeffective concentration of the peroxyacid make-up water 105 is combinedwith the concentrate from drum 104 into the tank 101. The peroxyacidsanitizer material in an appropriate concentration is passed through aheater 108 to reach a temperature of about 45-50° C. The heatedperoxyacid material is sprayed within sterilizing tunnel 102 into andonto all surfaces of the bottle 110. An intimate contact between theperoxyacid material and the bottle 110 is essential for reducingmicrobial populations to a sanitizing level. After contact with theperoxyacid material and after dumping any excess material from thebottles, the sterilized bottles 110 are then passed to a fresh waterrinse tunnel 103. Fresh water 108 is provided from a fresh water make-upinto a spray rinsing tunnel 103. Excess spray drains from rinsing tunnel103 to drain 106. Within the tunnel 103, sanitized bottles 110 a arethoroughly rinsed with fresh water. The complete removal of theperoxyacid material from the bottles 110 a is important for maintaininghigh quality of the beverage product. The rinsed sanitized bottles 110 bare then removed from the rinsing tunnel. The day tank 101, thesterilizing tunnel 102 and the rinsing tunnel 103 are all respectivelyvended to wet scrubber or vent 111 a, 111 b or 111 c to remove vapor orfumes from the system components. The sanitizer material that has beensprayed and drained from the bottles 110 a accumulate in the bottom ofthe spray tunnel 102 and is then recycled through recycle line andheater 107 into the day tank 101.

The day tank is used for mixing and storing the peroxyacid materialwhich can be a 0.1 to 5 wt %, preferably 2 to 4 wt % peroxyacetic acid.All active treating equipment should be vented to a wet scrubber toprevent peroxide peracetic acid or acetic acid fumes from entering theatmosphere from the treatment equipment. Draining of the containers oftheir peroxyacid contents is important to reduce carry over minimizedproduct loss. Water used to make up the active sanitizer material shouldbe deionized to maximize the useful life of the peroxyacid material.Deionized water maintains the highest effective concentration andreduces spotting and filming. The contact between the bottles and theperoxyacid material is typically at a temperature of greater than about40° C. In order to obtain sanitization of beverage containers at about700 ppm per acid to about 2500 ppm per acid contact at 40-60° C. for atleast 7 seconds contact time is required. Preferably, the sanitizationequipment, day tank, sanitizing tunnel and rinsing tunnel aremanufactured from polyolefin structural plastics, passivated stainlesssteel or other non-corrosion sensitive production materials.

In the cold aseptic filling of 16 ounce polyethylene terephthalate (PETbottle) beverage containers, a process has been adopted using aperoxyacid sanitizer or mixed peroxyacid sanitizer material. Theperoxyacid material is diluted to a concentration of about 0.1 to about3 wt % and is maintained at an effective elevated temperature of about40° C. to about 60° C., preferably about 50° C. The spray or flood ofthe bottle with the material ensures contact between the bottle and thesanitizer material for at least 7 seconds. After flooding is complete,the bottle is drained of all contents for a minimum of 2 secondsfollowed by a 5 second water rinse with sterilized water using about 200milliliters of water at 38° C. (100° F.). The bottle is then drained ofthe sterilized water rinse for at least 2 seconds and is immediatelyfilled with liquid beverage. After the rinse is complete, the bottlesmaintain less than 3 milliliters of rinse water after draining.

EXAMPLE 1

Experiments were conducted to determine the antimicrobial efficacy ofpure peroxyacids. Table I below demonstrates the antimicrobial efficacyof pure peroxyacids at very low levels when exposed to S. aureus and E.coli. The peroxyacids listed in Table I were tested by diluting them in0.05 M citrate buffer made in distilled water and were exposed to thebacteria for 30 seconds at 20° C. As Table I indicates, thediperoxyacids were somewhat less active than the peroxyfatty acids.Peroxydecanoic acid was very effective at very low levels against S.aureus, but higher levels were required to be effective against E. coli.Higher levels were also required at pH 5.

TABLE I Comparison of biocidal Activity of Peroxyacids Minimumconcentration required Peroxyacid for 5-log reduction (ppm)^((a)) pH S.aureus E. coli Peroxyhexanoic (C₆) 3.5 15 15 5.0 20 15 Diperoxyadipic(C₆) 3.5 >50 40 5.0 >60 35 Peroxyoctanoic (C₈) 3.5 5 5 5.0 10 15Peroxydecanoic (C₁₀) 3.5 3 10 5.0 1 30 Diperoxysebacic (C₁₀) 3.5 15 155.0 10 50 ^((a))Peroxyacids tested at 5-ppm increments, or at 1, 3, and5 ppm where appropriate.

In Table II below, the antimicrobial synergism between the C₂ and C₃peroxyacids when combined with C₈ and C₁₀ peroxyfatty acids is shown. AsTable II shows, there was little or no antimicrobial activity when theC₂ and C₃ peroxyacids and the C₈ and C₁₀ peroxyfatty acids were testedalone. However, when a C₂ or C₃ peroxyacid was combined with a C₈ or C₁₀peroxyfatty acid, the bacterial kill of E. coli multipliedexponentially. These tests were conducted at pH 4.5 or 5, the pH atwhich E. coli is more difficult to kill (see Table II).

TABLE II Synergistic Interaction of Peroxyacids C₂ C₃ C₈ C₁₀[Peroxyacetic] [Peroxypropionic] [Peroxyoctanoic] [Peroxydecanoic] (ppm)(ppm) (ppm) (ppm) Log Reduction 25 0   0^(a) 0 5 0.1^(a) 25 5 3.8^(a) 250 0.3^(b) 0 6 0.1^(b) 25 6 3.9^(b) 30 0 0.7^(a) 0 6   0^(a) 30 6 2.6^(a)^(a) E. coli, pH 5, distilled water ^(b) E. coli, pH 4.5, 500 ppm hardwater

EXAMPLE 2

A mixture of short chain fatty acids commercially available from EmeryCorporation under the designation “EMERY 658” was employed in producinga sanitizing concentrate composition of the present invention. The“EMERY 658” acid is a mixture of caprylic acid (C₈) and capric acid(C₁₀). The peroxyacids were prepared by the method of Parker, et al., J.Amer. Chem. Soc., 77, 4037 (1955) which is incorporated by reference.The peroxyacid component (also containing 34% acetic acid and 10%hydrogen peroxide) was combined with a pre-made solution of 10.42%peracetic acid, a separate amount of acetic acid, water, and ann-octanesulfonate hydrotzope coupler (NAS 8D). The final composition ofthis Example was as listed in Table III.

EXAMPLE 3

A second composition of the present invention was prepared as describedin Example 2, except that caprylic acid (C₈) and capric acid (C₁₀)replaced some of the peroxy acid of Example 2. The concentration ofperacetic acid was 5% while the concentration of peroxy acids wasreduced to 1.5% (See Table III).

EXAMPLE 4

The composition of Example 4 was prepared according to the procedure ofExample 2, except that no peracetic acid or hydrogen peroxide was addedto the composition. The acetic acid component was increased to 39 wt-%and the composition contained 5% peroxy acid (see Table III). Also, achelating agent (Dequest 2010) was added to the composition.

EXAMPLE 5

The composition of Example 5 was prepared the same as Example 4 exceptthat caprylic acid and capric acid were added to the composition inaddition to the percaprylic and percapric acid of Example 4. Thecomposition contained 3.5% fatty acid and 1.5% peroxy acid (see TableIII).

EXAMPLE 6

Example 6 was prepared with only peracetic acid, acetic acid, hydrogenperoxide, and water. No peroxy acids or fatty acids were added to thecomposition of Example 6. The concentration of total peroxyacid wasabout 5% and the acetic acid concentration was about 39% (see Table III).

EXAMPLE 7

Example 7 was prepared the same as Example 5 except that no peroxyacidswere employed, only a mixture of fatty acids and acetic acid was used,along with water, NAS BD, and Dequest 2010. The composition contained 5%fatty acid (see Table III).

TABLE III Wt-% of Ingredients Ingredient Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6Ex. 7 Peracetic Acid 50 50 — — 50 — (10.42% solution, 34% acetic acid,10% H₂O₂) Acetic Acid 22 22 39 39 22 39 Percaprylic Acid 3.75 1.125 3.751.125 — — (C₈) Percapric Acid 1.25 0.375 1.25 0.375 — — (C₁₀) CaprylicAcid — 2.625 — 2.625 — 3.75 (C₈) Capric Acid (C₁₀) — 0.875 — 0.875 —1.25 NAS 8D 10 10 10 10 — 10 Water 13 13 45 45 28 45 Dequest 2010 — — 11 — 1

Antimicrobial Efficacy of Examples 2-7

The compositions prepared according to Examples 2-7 were tested fortheir antimicrobial efficacy using the testing procedure of the standardA.O.A.C. sanitizing test. All of the samples tested of Examples 2-7 weremade about 1 hour prior to testing. The bacteria used in the testprocedure were S. aureus and E. coli. Distilled water was used to dilutethe concentrate compositions of Examples 2-7 and the composition wasemployed at room temperature. The following neutralizers were employedin the test: 0.1% thiosulfate, peptone, 0.5% K₂HPO₄, 0.025% catalase forperacetic acid; chambers for fatty acid; 0.1% thiosulfate, peptone,0.025% catalase for peracetic acid/fatty acid (peroxy acid). Theantimicrobial activity of Examples 2-7 are summarized in Table IV.Examples 2 and 3 were tested using four samples (a,b,c,d) and Examples4-7 were tested using two samples (a,b). As can be seen in Table IV,Examples 25 exhibited excellent kill (>log 6) of both S. aureus and E.coli at 50 ppm of peroxyacid. Examples 6 and 7 (containing no peroxyacids) exhibited little or no activity. More specifically, Example 2 wastested at 1,000 and 500 ppm total product (50 and 25 ppm of bothperoxyacetic acid and peroxy acid). At these low concentrations, theperoxyacid combination gave a 6-7 log reduction in the bacterial count.Example 3 was tested at 1,000 and 500 ppm total product, and also had a6-7 log reduction in the bacterial count. At the 500 ppm productconcentration the product corresponds to 25 ppm of peroxyacetic acid and7.5 ppm of peroxy acids. Example 4, at 1,000 ppm of total product (50ppm of peroxy acid), completely killed all bacteria (greater than 7 logreduction). Example 5 also resulted in a complete kill using 1,000 ppmof total product (15 ppm peroxy acid). Example 6 contained no peroxyacid (only 50 ppm of peroxyacetic acid) and showed no activity againstS. aureus and poor activity against E. coli. This is due to the factthat peroxyacetic acid is generally not effective at this level, and isgenerally used at concentrations greater than 100 ppm. Example 7,containing 5% fatty acid (30 ppm) and no peroxy acid at 1,000 ppm totalproduct showed no activity toward either organism.

TABLE IV Mixed Acid Test Product [POAA¹/POA²/FA³] Log₁₀ Kill Sam-Concentration Concentration S. E. Ex. ple (ppm) (ppm) pH aureus coli 2 a1000 50/50/0 3.5 6.13 >7.30 b 1000 50/50/0 3.5 6.52 7.30 c  500 25/25/03.68 6.63 7.00 d  500 25/25/0 3.68 6.78 7.30 3 a 1000 50/15/35 3.52 7.187.30 b 1000 50/15/35 3.52 6.63 6.90 c  500 25/7.5/17.5 3.68 6.70 6.76 d 500 25/7.5/17.5 3.68 7.18 7.00 4 a 1000 0/50/0 3.5 >7.18 >7.30 b 10000/50/0 3.5 >7.18 >7.30 5 a 1000 0/15/35 3.5 >7.18 >7.30 b 1000 0/15/353.5 >7.18 >7.30 6 a 1000 50/0/0 3.49 NMA⁴ 3.48 b 1000 50/0/0 3.49 NMA3.80 7 a 1000 0/0/30 3.46 NMA NMA b 1000 0/0/30 3.46 NMA NMA ¹POAA =Peroxyacetic Acid ²POA Peroxy Acid ³FA = Fatty Acid ⁴NMA = No measurableactivity

EXAMPLE 8-11

Examples 8-11 were prepared by substantially the same procedure as theprevious Examples, except that hydrogen peroxide (H₂O₂) was mixed withacetic acid and C₈-₁₀ fatty acids (Emery 658) to make the peroxyacids ofthe composition, Table V summarizes the components and amounts of thevarious compositions of Examples 8-11 which were made.

TABLE V Peroxyacid Test Formulations Ingredient Ex. 8 Ex. 9 Ex. 10 Ex.11 Acetic Acid 44 39 34 49 H₂O₂ (35%) 40 40 40 40 Dequest-L 2010 1 1 1 1NAS 8D 10 10 10 10 Emery 658 5 10 15 —

Peroxyacid Stability, Biocidal Activity of Examples 8-11

Each of Examples 8-11 were tested for peroxyacid stability and biocidalactivity using the A.O.A.C. sanitizing test against S. aureus and E.coli at room temperature with the formulations diluted in distilledwater. Tables VI-IX show the biocidal activity of each formulation.Generally all of the formulations reached maximum peroxyacid formationwithin about 12 days. All of the formulations obtained about 12.5%peroxyacid except Example 10 (15% fatty acid) which obtained about 11.5%peroxyacid.

Table VI summarizes the biocidal activity of Example 8 in which thecomposition was measured for biocidal activity on the first day up today 33. At 250 ppm of total product, there were about 4-5 ppm of peroxyacid and about 20 ppm of peracetic acid as determined by carbon 13 NMRspectroscopy. The results are summarized in Table VI.

TABLE VI Peroxyacid Stability, Biocidal Activity of Example 8 ReductionPeroxyacid Test^((a)) Test Ave. Log Day Percent Concentration pH S.aureus E. coli 1 4.28 250 ppm 3.92 6.28 NMA^((b)) 6 11.00 250 ppm3.91 >7.38 >7.18 8 11.08 250 ppm 3.86 >7.11 >7.12 12 12.43 250 ppm3.83 >7.18 6.96 15 12.74 250 ppm 3.88 6.83 — 33 10.18 250 ppm 3.83 5.186.34 ^((a))ppm total product ^((b))No measurable activity

The biocidal activity of Example 9 is summarized in Table VII below. Theperacetic acid concentration at 250 ppm of product was about 20-21 ppmand the concentration of peroxy acid was about 11 ppm. The concentrationof peracetic acid at 50 ppm of product was about 4 ppm and theconcentration of peroxy acid was about 2 ppm.

TABLE VII Peroxyacid Stability Biocidal Activity of Example 9 ReductionPeroxyacid Test^((a)) Test Ave. Log Day Percent Concentration pH S.aureus E. coli 1 4.88 250 ppm 3.95 >7.60 NMA^((b)) 6 10.62 250 ppm3.92 >7.38 >7.18 8 11.61 250 ppm 3.98 >7.11 >7.12 12 12.47 250 ppm3.91 >7.18 >7.23 15 12.00 250 ppm 3.95 6.95 — 120 ppm 4.18 >7.13 —  50ppm 4.41 6.39 — 33 10.49 250 ppm 3.85 5.20 6.22 ^((a))ppm total product^((b))No measurable activity

The biocidal activity of Example 10 is summarized in Table VIII below.At 250 ppm of product the peracetic acid concentration was about 19 ppmand the peroxy acid concentration was about 14 ppm.

TABLE VIII Peroxyacid Stability Biocidal Activity of Example 10Reduction Peroxyacid Test^((a)) Test Ave. Log Day Percent ConcentrationpH S. aureus E. coli 1 4.84 250 ppm 3.90 >7.60 NMA^((b)), 4.04 6 9.81250 ppm 3.96 >7.38 >7.18 8 10.99 250 ppm 3.96 >7.11 >7.12 12 11.47 250ppm 3.94 >7.18 >7.23 15 11.48 250 ppm 3.96 6.83 — 33 10.49 250 ppm 3.955.25 6.53 ^((a))ppm total product ^((b))No measurable activity

The biocidal activity of Example 11 is summarized in Table IX below. At250 ppm of product there was about 27 ppm of peracetic acid. At 1000 ppmof product there was about 108 ppm of peracetic acid. No fatty acid wasemployed in the composition of Example 11.

TABLE IX Biocidal Activity of Example 11 Reduction Peroxyacid Test^((a))Test Ave. Log Day Percent Concentration pH S. aureus E. coli 5 10.95 250 ppm 3.90 NMA^((b)) NMA 7 12.03 1000 ppm 3.50 4.60 >7.12 11 12.441000 ppm 3.49 6.38 6.64 14 12.53 1000 ppm 3.50 4.17 — 32 10.77 1000 ppm3.45 4.77 6.44 ^((a))ppm total product ^((b))No measurable activity

When comparing the formulations containing fatty acid (Tables VI-VIII),poor activity was measured against E. coli one day after beingformulated. Since the total peroxyacid values were low, more fatty acidwas present and gram negative bacteria tend to be less sensitive thangram positive bacteria to the C₈-C₁₀ fatty acids. However, as moreperoxyacid developed over the days indicated, increased biocidalactivity against E. coli was observed. Table IX indicates that to obtainacceptable activity (greater than or equal to 5 log reduction) usingonly peracetic acid, the peracetic acid must be tested over 100 ppmactive. Secondly, this oxidizing compound is more effective against E.coli than S. aureus.

Generally all the formulations containing fatty acid remain stable afterabout 1 month. This was confirmed by repeated testing over time at 250ppm total product for each formulation in which greater than 5 logreductions were measured against S. Aureus and E. coli.

EXAMPLES 12-17

The biocidal activity of a two-component system containing bothperacetic acid and fatty acid was investigated using the A.O.A.C.sanitizing test. Table X shows the product formulations examined. Thetest samples include controls showing biocidal activity of NAS 8D aswell as fatty acid kill against S. aureus. All the samples were testedin distilled water.

TABLE X Wt-% Ingredient Ingredient Ex. 12 Ex. 13 Ex. 14 Ex. 15 Ex. 16Ex. 17 Base 1^((a)) 80 80 80 80 — — Base 2^((b)) — — — — 80 80 NAS 8D 10— 10 10 10 10 Octanoic Acid — — 10 — — 10 Emery 658 — — — 10 10 — H₂O 1020 — — — — ^((a))H₂O₂, 35%; acetic acid, 35%; Dequest 2010, 1%; H₃PO₄(85%), 29%. ^((b))Acetic acid, 35%; Dequest 2010, 1%; H₃PO₄ (85%), 29%;H₂O, 35%.

Table XI shows the activity measurement of each of Examples 12-17 atvarious test concentrations. When testing the peracetic acid formulationof Examples 12 and 13 (having no fatty acid), biocidal activity occurredonly at 100 ppm or greater. Biocidal activity (greater than 4 logreduction) was measured at a minimal concentration of 10 ppm peroxyacidwith fatty acid in the system (Example 14). At 10 ppm peroxyacid, thecomposition containing Emery 658 (Example 15) had better activity thanthe system containing only C₈ (octanoic) fatty acid (Example 14). In thefatty acid controls (Examples 16 and 17), the Emery 658 had morebiocidal activity than the C₈ fatty acid. At total product testconcentrations equivalent to 10 or 25 ppm peroxyacid, the fatty acid inthe system of Example 16 did not have significant biocidal activity.Example 17 did not have significant biocidal activity at any testconcentration.

TABLE XI Peroxyacid Biocidal Activity Against S. aureus Peroxyacid %Concentration Test Example Reduction (ppm Peroxyacid) pH Log^((a)) 127.02  50 2.79 NMA^((b)) 100 2.54 5.45 150 2.41 >7.70 13 6.25  50 2.76NMA 100 2.52 4.51 150 2.40 5.84 14 9.32  10 3.52 4.22  25 3.16 >7.70  502.90 >7.70 15 9.73  10 3.50 6.82  25 3.19 7.55  50 2.88 >7.70 16 ——^((c)) 3.53 0.70 —^((c-1)) 3.18 1.04 —^((c-2)) 2.88 4.07 17 — —^((d))3.51 0.93 —^((d-1)) — 0.66 —^((d-2)) — 0.97 ^((a))Average of duplicatetesting. ^((b))No measurable activity. ^((c))Same total productconcentrations as Example 15 @10 ppm peroxyacid (about 100 ppm product).^((c-1))Same total product concentration as Example 15 @25 ppmperoxyacid (about 250 ppm product). ^((c-2))Same total productconcentration as Example 15 @50 ppm peroxyacid (about 500 ppm product).^((d))Same total product concentration as Example 14 @10 ppm peroxyacid(about 100 ppm product). ^((d-1))Same total product concentration asExample 14 @25 ppm peroxyacid (about 250 ppm product). ^((d-2))Sametotal product concentration as Example 14 @50 ppm peroxyacid (about 500ppm product).

The biocidal activity of a peracetic acid/fatty acid system was measuredcomparing freshly made formulations to month-old formulations ofExamples 14 and 15. These formulations are shown in Table XII whichcompares the titration values of month-old formulations to the samefreshly prepared. Table XIII shows the biocidal activity of month-oldand fresh formulations of Examples 14 and 15.

TABLE XII Peroxyacid Titration Values Ex. 14 Ex. 15 Ex. 14 Ex. 15 Dateformulated Month-Old Month-Old Fresh Fresh % H₂O₂ 2.15 2.07 1.99 1.99 %Peroxyacid 5.37 5.35 4.85 4.86 % Total O₂ 2.14 2.10 1.96 1.96

TABLE XIII Peroxyacid Biocidal Activity Against S. aureus Peroxyacid (%)Test Concentration Test Example Reduction (ppm Peroxyacid) pH Log^((a))14 5.37 10 3.46 NMA^((b)) (Month-Old) 25 3.07 >7.48 14 4.85 10 3.34 5.07(Fresh) 25 2.97 7.30 15 5.35 10 3.52 5.29 (Month-Old) 25 3.04 7.24 154.86 10 3.42 NMA^((c))/ 3.68 (Fresh) 25 2.99 7.48 ^((a))Average ofduplicate testing. ^((b))No measurable activity. ^((c))Duplicate testingin which only one sample exhibited biocidal activity.

As can be seen from Table XIII, biocidal activity in the peraceticacid/fatty acid system occurs at test concentrations as low as 10 or 25ppm peroxyacid. Mixed results occurred at 10 ppm peroxyacid between themonth-old and fresh formulations of Examples 14 and 15, however,increasing the concentration to 25 ppm resulted in a uniform killactivity (>7 log reduction).

An additional test was run to determine how quickly compounds exhibitingbiocidal activity are formed upon adding fatty acid to a peracetic acidsystem. Examples 12, 15 and 16 were tested. Examples 12 and 15 wereformulated the day before testing and were day-old samples. Another testsample of Example 15 was formulated immediately prior to testing.Example 16 containing Base 2 (no H₂O₂) was used to show biocidalactivity from the fatty acid at low test concentrations. Table XIV showsthe biocidal activity of each Example in distilled water against S.aureus.

TABLE XIV Biocidal Activity Against S. aureus ppm Test Log^((a)) ExampleAge Peroxyacid pH Reduction 12 1 day 50 2.94 NMA^((b)) 100  2.71 6.60 151 day 10 3.68 7.02 25 3.35 >7.20 15 fresh 10 3.76 NMA 25 3.32 NMA 16 22days —^((c)) 3.74 NMA —^((d)) — NMA ^((a))Average of duplicate testing.^((b))No measurable activity. ^((c))Equivalent total productconcentration as Example 15 (day old) @ 10 ppm peroxyacid.^((d))Equivalent total product concentration as Example 15 (day old) @25 ppm peroxyacid.

The data from Table XIV suggests that the formation of compoundscontaining biocidal activity when adding fatty acid to a peracetic acidsystem is not immediate, but does occur within a day. The formation ofcompounds exhibiting biocidal activity occurred within a day afteradding fatty acid to the peracetic acid system as in Example 15 withbiocidal activity occurring at a concentration as low as 10 ppmperoxyacid. Thus, the biocidal activity is not due to the merecombination of fatty acid and peroxyacetic acid, but the fatty acid mustbe converted to the peroxy acid before substantially enhanced biocidalactivity occurs.

EXAMPLES 18-22

A two-component system containing peracetic acid and peroxy acid wasformulated and tested to determine its sanitizing activity over just aperacetic acid system. Table XV shows premixes 1 and 2 used in makingthe composition. The premixes were both made with H₂O₂ (35% solution),acetic acid, Dequest 2010, and with/without H₃PO₄. Premix 1 was madeabout 5 months before premix 2. To each premix was added NAS 8D, a C₈fatty acid or Emery 658 as shown in Table XVI to complete theformulation of Examples 18-21. Example 22 was formulated as a controland had no fatty acid.

TABLE XV Peroxyacid Premixes Wt-% Component Component Premix 1 Premix 2H₂O₂ (35%) 75.0 35.0 Acetic acid (glacial) 24.0 35.0 Dequest 2010  1.0 1.0 H₃PO₄ (85%) — 29.0

TABLE XVI Peroxy Acid/Peracetic Acid Formulations Wt-% Ingredient(Control) Ingredient Ex. 18 Ex. 19 Ex. 20 Ex. 21 Ex. 22 Premix 1 80.0 —80.0 — — Premix 2 — 80.0 — 80.0 — NAS 8D 10.0 10.0 10.0 10.0 — C₈ FattyAcid 10.0 10.0 — — — Emery 658 — — 10.0 10.0 — Acetic Acid — — — — 24.0(Glacial) H₂O₂ (35%) — — — — 75.0 Dequest 2010 — — — —  1.0

Table XVII shows the sanitizing activity measured from each formulationof Examples 18-22 at 50, 100, or 150 ppm peracetic acid against S.aureus.

TABLE XVII Sanitizing Efficacy of Peroxy Acid/ Peracetic Acid System vs.Sanitizing Efficacy of Peracetic Acid System Total Peroxyacid^((a))Fatty Test (Percent) Acid Concentration Test Example Reduction (Percent)(ppm) pH Log^((b)) 18 7.69 10.0 150 3.53 >7.06 100 3.64 >7.06 503.83 >7.06 19 11.21 10.0 150 2.71 >7.06 100 2.80 >7.06 50 3.08 >7.06 209.08 10.0 150 3.64 >7.06 100 3.65 >7.06 50 3.85 >7.06 21 10.92 10.0 1502.68 >7.06 100 2.77 >7.06 50 3.10 >7.06 22 10.40 — 150 3.56  7.06(Control) 100 3.68  3.89 50 3.93 NMA^((c)) ^((a))As peracetic acid^((b))Average of duplicate testing against S. aureus. ^((c))Nomeasurable activity.

Extremely good kill (>7 log reduction) was obtained with or withoutH₃PO₄ in the peroxy acid formulations of Examples 18-21. The twocomponent system of C₈ fatty acid or Emery 658 in combination withperacetic acid (Examples 18-21) had significantly better kill than theperacetic acid system alone (Example 22) at a test concentration of 50to 100 ppm. No activity was measured at 50 ppm with the single peraceticacid system of Example 22.

EXAMPLES 23-26

The effect of alkyl chain length on antimicrobial efficacy of peroxyacids was determined for percaprylic (C₈) acid, percapric (C₁₀) acid anda percaprylic/percapric (3:1) perfatty acid mixture using thecompositions of Examples 23-26 summarized in Table XVIII below.

TABLE XVIII Wt-% of Ingredient Ingredient Ex. 23 Ex. 24 Ex. 25 Ex. 26Percaprylic  1 — — — (C₈) Acid Percapric —  1 — — (C₁₀) Acid C₈ + C₁₀(3:1) — —  1 — Perfatty Acid Acetic Acid 10 10 10 10 Water 84 84 84 85NAS 8D  5  5  5  5

The antimicrobial efficacy of Examples 23-26 are summarized in Table XIXbelow. Examples 23-25 were tested using three samples (a, b, c) of 5,10, and 15 ppm of perfatty acid respectively. Example 26, containing noperfatty acid, was diluted to an equivalent formulation of Examples23-25 containing perfatty acid. As can be seen from Table XIX,significant kill occurred at 5 ppm for S. aureus using Examples 23-25.Significant kill occurred against E. coli at 10 ppm of perfatty acid inExamples 23-25.

TABLE XIX Antimicrobial Efficacy of Examples 23-26¹ Perfatty AcidConcentration Log Kill Example Sample (ppm) S. aureus E. coli 23 a 5 >7.0 3.6 b 10 — >7.2 c 15 — >7.2 24 a  5 >7.0 3.0 b 10 — >7.2 c 15— >7.2 25 a  5 >7.0 <3.0 b 10 — >7.2, 5.5 c 15 — >7.2 26 a —^(a) 0 — b—^(b) — 0 ^(a)- Equivalent total product concentration as Examples 23,24, 25 at 5 ppm perfatty acid. ^(b)- Equivalent total productconcentration as Examples 23, 24, 25 at 15 ppm perfatty acid. ¹Example26 (having no perfatty acid) did not produce any kill of eithermicroorganism.

EXAMPLE 27

The antimicrobial activity of percaprylic acid against E. coli wasmeasured at a 30 second exposure at varying pH's. The formulationcontained 94% water, 5% NAS 8D, and 1% percaprylic acid. The formulationwas diluted in a buffer of 0.05 M citrate and 0.05 M potassiumphosphate. The log kill of this formulation at increasing pH's issummarized in Table XX. Samples containing 7 ppm and 25 ppm ofpercaprylic acid were tested. As Table XX indicates, significant kill at7 ppm occurred at a pH of 3.0. Significant kill levels were maintainedat 25 ppm through a pH of 7.0.

TABLE XX Antimicrobial Efficacy of Percaprylic Acid against E. coli LogKill Log Kill (Perfatty (Perfatty Concentration Concentration pH 7 ppm)25 ppm) 3.0 >7.2 >7.2 5.0 <3.0 >7.2 7.0 <3.0 >7.2 8.9 — <3.0 9.0 <3.0 —

EXAMPLES 28-30

The compositions of Examples 28-30 were made to determine thelimitations on biocidal activity of compositions containing at least 30%acetic acid. Higher acetic acid formulations were also tested for theirbiocidal activity. The composition of Example 30 was prepared with nocoupler (NAS 8D). The compositional ingredients of Examples 28-30 aresummarized below in Table XXI.

TABLE XXI Wt-%-of Ingredient Ingredient Example 28 Example 29 Example 30Acetic Acid 30.0 50.0 50.0 H₂O₂ (35%) 30.0 15.0 15.0 Dequest 2010  1.0 1.0  1.0 C₈ Fatty Acid  4.0  6.0  5.0 NAS 8D  5.0  5.0 — (Spray Dried)Distilled Water 30.0 23.0 29.0

The antimicrobial efficacy of Examples 28-30 was determined using theprocedure of the standard A.O.A.C. sanitizing test. The compositions ofExamples 28-30 were diluted with 500 ppm hard water and employed at 25°C. The bacteria used in the test procedure were S. aureus and E. coli,and a TGE plating medium was employed. Exposure time of the compositionsto the bacteria was 30 seconds. The neutralizer employed in the testingprocedure contained 0.1% thiosulfate, 1.0% peptone, and 0.025% catalase.The antimicrobial activity of Examples 28-30 is summarized in Table XXIIbelow.

TABLE XXII Biocidal Activity of Examples 28-30 Log Reduction FormulationConcentration pH S. aureus E. coli Example 28 1 oz: 8 gal.^(a)4.48 >7.15 >6.89 1 oz: 10 gal.^(b) 4.83 >7.15 >6.89 1 oz: 12 gal.^(c)5.04 >7.15 6.41 1 oz: 14 gal.^(d) 5.52 >7.15 5.76 1 oz: 16 gal.^(e)5.94 >7.15 2.95 Example 29 40 ppm Active 4.16 >7.15 >6.89 Example 30 40ppm Active 4.04 >7.15 >6.89 ^(a)54.2 ppm peroxyacid ^(b)43.3 ppmperoxyacid ^(c)36.1 ppm peroxyacid ^(d)31.0 ppm peroxyacid ^(e)27.2 ppmperoxyacid

As Table XXII indicates, very low concentrations of combinations ofperoxyacetic acid and peroxyfatty acid are very effective in killingbacteria. Also, Example 30 showed that the composition of the inventionis antimicrobially effective without a hydrotrope coupler.

EXAMPLE A

RAW MATERIAL WT PERCENT Water, deionized 26.00 Hydroxyethylidene-1,1- 1.50 diphosphonic acid (60% aqueous) Hydrogen peroxide (35% aqueous)26.00 Acetic acid, 100% 30.00 Octane sulfonate 12.50 Octanoic acid  4.00TOTAL 100.00 

Objective

The objective of this analysis was to determine the antimicrobialefficacy of Example A when prepared in 80 ppm and 500 ppm synthetic hardwater at a concentration of 2.2% against Chaetomium bostrychodesascospores (Coca-Cola Japan mold isolate), utilizing exposure times of30 seconds, 1.0 minute, 2.0 minutes and 5.0 minutes.

Method Parameters

Test mL of Substance Test mL of Name Diluent Concentration SubstanceDiluent Example A  80 ppm 9.2% 22 978.0 synthetic hard water as CaCO₃Example A 500 ppm 2.2% 22 978.0 synthetic hard water as CaCO₃

Test System: Chaetomium bostrychodes ascospores (mold contaminant fromCoca-Cola Japan)

Test Temperatures: 40° C. and 50° C.

Exposure Times: 30 seconds, 1.0, 2.0 and 5.0 minutes

Neutralizer: 1% Sodium thiosulfate

Plating Medium: Potato Dextrose Agar (PDA)

Incubation: 5-7 days at 26° C.

Results

Control Numbers (CFU/mL) Test System A B C Average Chaetomium  12 × 10⁶  9 × 10⁶  14 × 10⁶ 1.2 × 10⁷ bostrychodes 30 1.0 2.0 5.0 Test SecondMinute Minute Minute Substance Exposure Exposure Exposure ExposureChaetomium bostrychodes Average Survivors (CFU/mL) at 40° C. Example A5.5 × 10⁶ 6.4 × 10⁶ 6.0 × 10⁶ 1.5 × 10⁴  (80 ppm synthetic hard water)Example A 5.6 × 10⁶ 5.9 × 10⁶ 5.9 × 10⁶ 2.3 × 10⁴ (500 ppm synthetichard water) Chaetomium bostrychodes Average Survivors (CFU/mL) at 50° C.Example A 4.9 × 10⁶ 2.0 × 10⁶ <10 <10  (80 ppm synthetic hard water)Example A 5.5 × 10⁶ 3.0 × 10⁶ <10 <10 (500 ppm synthetic hard water)Chaetomium bostrychodes Log Reductions at 40° C. Example A 0.34 0.270.30 2.90  (80 ppm synthetic hard water) Example A 0.33 0.31 0.31 2.72(500 ppm synthetic hard water) Chaetomium bostrychodes Log Reductions at50° C. Example A 0.39 0.78 >6.08 >6.08  (80 ppm synthetic hard water)Example A 0.34 0.60 >6.08 >6.08 (500 ppm synthetic hard water)

Conclusions

Example A at 2.2% when diluted in both 80 ppm and 500 ppm synthetic hardwater achieved a >6.08 log reduction (no survivors) after a 2.0 minuteexposure time at 50° C., while achieving a <1.00 log reduction at 30seconds and 1.0 minute. At 40° C., Example A achieved a 2.90 (80 ppmsynthetic hard water) and a 2.72 (500 ppm synthetic hard water) logreduction after a 5.0 minute exposure time, while achieving a <1.00 logreduction at 30 seconds, 1.0 minute and 2.0 minutes. To achieve totalkill of Chaetomium bostrychodes ascospores, Example A should be used ata concentration of 2.2% at 50° C., utilizing an exposure time of 2.0minutes.

Example B

RAW MATERIAL WT % GM/1100 Acetic Acid 78.00 858.00 Hydrogen Peroxide 35%21.00 231.00 Phosphonate sequestrant  1.00  11.00 TOTAL 100.00  1100.00 

Objective

The objective of this analysis was to determine the antimicrobialefficacy of Example B and Example C when prepared in 80 ppm and 500 ppmsynthetic hard water at concentrations of 0.85% Example B and 0.05%Example C against Chaetomium bostrychodes ascospores (Coca-Cola Japanmold isolate), utilizing exposure times of 30 seconds, 1.0 minute, 2.0minutes and 5.0 minutes.

Method Parameters

Test mL of Substance Test mL of Name Diluent Concentration SubstanceDiluent Example B  80 ppm 0.85% 8.5 991.5 synthetic hard water as CaCO₃Example B 500 ppm 0.85% 8.5 991.5 synthetic hard water as CaCO₃ ExampleC  80 ppm 0.05% 0.5 999.5 synthetic hard water as CaCO₃ Example C 500ppm 0.05% 0.5 999.5 synthetic hard water as CaCO₃

Test System: Chaetomiumn bostrychodes ascospores (mold contaminant fromCoca-Cola Japan)

Test Temperatures: 40° C. and 50° C.

Exposure Times: 30 seconds, 1.0, 2.0 and 5.0 minutes

Neutralizer: 1% Sodium thiosulfate

Plating Medium: Potato Dextrose Agar (PDA)

Incubation: 5-7 days at 26° C.

Results

Control Numbers (CFU/mL) Test System A B C Average Chaetomium   5 × 10⁴  4 × 10⁴   7 × 10⁴ 5.3 × 10⁴ bostrychodes 30 1.0 2.0 5.0 Test SecondMinute Minute Minute Substance Exposure Exposure Exposure ExposureChaetomium bostrychodes Average Survivors (CFU/mL) at 40° C. Example B1.1 × 10⁴ 1.7 × 10⁴ 1.9 × 10⁴ 1.2 × 10⁴  (80 ppm synthetic hard water)Example B 1.8 × 10⁴ 2.2 × 10⁴ 1.4 × 10⁴ 1.4 × 10⁴ (500 ppm synthetichard water) Example C 1.4 × 10⁴ 1.9 × 10⁴ 1.2 × 10⁴ 1.5 × 10⁴  (80 ppmsynthetic hard water) Example C 1.7 × 10⁴ 2.1 × 10⁴ 2.1 × 10⁴ 1.6 × 10⁴(500 ppm synthetic hard water) Chaetomium bostrychodes Average Survivors(CFU/mL) at 50° C. Example B 1.6 × 10⁴ 1.7 × 10⁴ 2.5 × 10³ <10  (80 ppmsynthetic hard water) Example B 1.5 × 10⁴ 1.4 × 10⁴ 5.0 × 10³ <10 (500ppm synthetic hard water) Example C 1.1 × 10⁴ 1.6 × 10⁴ 1.5 × 10⁴ 1.3 ×10⁴  (80 ppm synthetic hard water) Example C 1.8 × 10⁴ 2.9 × 10⁴ 2.5 ×10⁴ 2.1 × 10⁴ (500 ppm synthetic hard water) Chaetomium bostrychodes LogReductions at 40° C. Example B 0.68 0.49 0.44 0.65  (80 ppm synthetichard water) Example B 0.47 0.38 0.58 0.58 (500 ppm synthetic hard water)Example C 0.58 0.44 0.65 0.55  (80 ppm synthetic hard water) Example C0.49 0.40 0.40 0.52 (500 ppm synthetic hard water) Chaetomiumbostrychodes Log Reductions at 50° C. Example B 0.52 0.49 1.33 >3.72 (80 ppm synthetic hard water) Example B 0.55 0.58 1.03 >3.72 (500 ppmsynthetic hard water) Example C 0.68 0.52 0.55 0.61  (80 ppm synthetichard water) Example C 0.47 0.26 0.33 0.40 (500 ppm synthetic hard water)

Conclusions

Example B at 0.85% when diluted in both 80 ppm and 500 ppm synthetichard water achieved a >3.72 log reduction after a 5.0 minute exposuretime at 50° C., while achieving a <1.50 log reduction at 30 seconds, 1.0minute and 2.0 minutes. At 40° C., Example B achieved a <1.00 logreduction at all exposure times.

Example C achieved a <1.00 log reduction when diluted in both 80 ppm and500 ppm synthetic hard water at both 40° C. and 50° C. at all exposuretimes. Example C was prepared at 0.05%, which is the highestconcentration possible to obtain solubility. The percent peracetic acidin Example C is 10.16, while Example C contains 11.4 percent. Althoughthe concentrations of peracetic acid in both products are similar,Example C can be used at a much higher use dilution due to the absenceof octanoic acid in the formula.

Example C performed similarly to Example A at 50° C. after a 5.0 minuteexposure time, while at 40° C. Example A demonstrated better efficacy byachieving approximately a 2.60 log reduction after a 5.0 minute exposuretime.

Example C

RAW MATERIAL WT % Acetic Acid (100%) 54 H₂O₂ (35% aqueous) 30 Octanoicacid 15 Phosphonate sequestrant 1 TOTAL 100.00

Objective

The objective of this analysis was to determine the antimicrobialefficacy of Example D and Example A when prepared in synthetic hardwater 500 ppm as CaCO₃ at concentrations of 1.0% Example A and 0.175%Example A against Chaetomium bostrychodes utilizing exposure times of 30seconds, 1.0 minute, 2.0 minutes, 5.0 minutes and 10.0 minutes.

Method Parameters

Test mL of Substance Test mL of Name Diluent Concentration SubstanceDiluent Example D 500 ppm 1.0%  5.0  495.0  synthetic hard water asCaCO₃ Example A 500 ppm 0.75% 3.75 496.25 synthetic hard water as CaCO₃

Test System: Chaetomium bostrychodes ascospores (mold contaminant fromCoca-Cola Japan)

Test Temperatures: 25° C. and 40° C.

Exposure Times: 30 seconds, 1.0, 2.0, 5.0 and 10.0 minutes

Neutralizer: 1% Sodium thiosulfate/1% Peptone/0.025% Catalase

Plating Medium: Potato Dextrose Agar (PDA)

Incubation: 5-7 days (or until growth is visible at 26° C.

Results

Inocuium Numbers (CFU/mL) Test System A B C Average Chaetomium 27 × 10⁶31 × 10⁶ 30 × 10⁶ 2.9 × 10⁷ bostrychodes Chaetomium bostrychodesSurvivors (CFU/mL) Test 30 Second 1.0 Minute 2.0 Minute 5.0 Minute 10.0Minute Substance Exposure Exposure Exposure Exposure Exposure Example Aat 1.2 × 10⁵ 1.3 × 10⁵ 9.4 × 10⁴ 8.6 × 10⁴ 8.7 × 10⁴ 25° C. Example D at1.6 × 10⁵ 9.7 × 10⁴ 8.7 × 10⁴ 8.6 × 10⁴ 7.8 × 10⁴ 25° C. Example A at1.7 × 10⁵ 1.7 × 10⁵ 9.7 × 10⁴ 1.0 × 10⁵ 9.1 × 10⁴ 40° C. Example D at1.5 × 10⁵ 1.2 × 10⁵ 1.1 × 10⁵ 9.6 × 10⁴ 1.0 × 10⁵ 40° C. Chaetomiumbostrychodes Log Reduction Test 30 Second 1.0 Minute 2.0 Minute 5.0Minute 10.0 Minute Substance Exposure Exposure Exposure ExposureExposure Example A at 2.38 2.35 2.49 2.53 2.52 25° C. Example D at 2.262.48 2.52 2.53 2.57 25° C. Example A at 2.23 2.23 2.48 2.46 2.50 40° C.Example D at 2.32 2.38 2.42 2.48 2.46 40° C.

Conclusions

The results of this analysis demonstrated that at 25° C., Example D andExample A were similar in efficacy against Chaetomium bostrychodes.Example A at the 30 second exposure time achieved a log reduction of2.38, while Example D achieved a log reduction of 2.26. Example A andExample D were relatively static at subsequent exposure times. Example Aat 40° C. achieved a log reduction of 2.23 at the 30 second exposuretime, while Example D had a 2.32 log reduction at the 30 second exposuretime. Example A and Example D both were static at subsequent exposuretimes. Further testing will be performed using 2.2% Example A and 4.0%Example D in 500 ppm synthetic hard water as CaCO₃ and deionized water.

Ecample D

RAW MATERIAL WT % Hydrogen peroxide, 35% aqueous technical) 82.0 AceticAcid, 100% 10.2 Hydroxyethylidene-1,1-diphosphonic acid  1.0 (60%aqueous) (Dequest 2010) Deionized Water  6.8 TOTAL 100.00

Objective

The objective of this analysis was to determine the antimicrobialefficacy of Example A when prepared in 80 ppm and 500 ppm synthetic hardwater at a concentration of 2.2% against Arthrinium sacchari ascospores(Coca-Cola Japan mold isolate), utilizing exposure times of 30 seconds,1.0 minute, 2.0 minutes and 5.0 minutes.

Method Parameters

Test mL of Substance Test mL of Name Diluent Concentration SubstanceDiluent Example A  80 ppm 2.2% 22.0 978.0 synthetic hard water as CaCO₃Example A 500 ppm 2.2% 22.0 978.0 synthetic hard water as CaCO₃

Test System: Arthrinium sacchari ascospores (mold contaminant fromCoca-Cola Japan)

Test Temperatures: 40° C. and 50° C.

Exposure Times: 30 seconds, 1.0, 2.0, and 5.0

Neutralizer: 1% Sodium thiosulfate

Plating Medium: Potato Dextrose Agar (PDA)

Incubation: 5-7 days 26° C.

Results

Control Numbers (CFU/mL) Test System A B C Average Arthrinium   5 × 10⁷ 12 × 10⁷  10 × 10⁷ 9.0 × 10⁷ sacchari 1.0 2.0 5.0 Test 30 Second MinuteMinute Minute Substance Exposure Exposure Exposure Exposure Arthriniumsacchari Average Survivors (CFU/mL) at 40° C. Example A 2.5 × 10⁵ 5.0 ×10⁵ 7.5 × 10⁵ 5.5 × 10⁵  (80 ppm synthetic hard water) Example A 2.5 ×10⁵ 8.0 × 10⁵ 1.2 × 10⁶ 5.0 × 10⁵ (500 ppm synthetic hard water)Arthrinium sacchari Average Survivors (CFU/mL) at 50° C. Example A 6.5 ×10⁵ <10 <10 <10  (80 ppm synthetic hard water) Example A 2.5 × 10⁵ <10<10 <10 (500 ppm synthetic hard water) Arthrinium sacchari LogReductions at 40° C. Example A 2.56 2.26 2.08 2.21  (80 ppm synthetichard water) Example A 2.56 2.05 1.88 2.26 (500 ppm synthetic hard water)Arthrinium sacchari Log Reductions at 50° C. Example A2.14 >6.95 >6.95 >6.95  (80 ppm synthetic hard water) Example A2.56 >6.95 >6.95 >6.95 (500 ppm synthetic hard water)

Conclusions

Example A at 2.2% when diluted in both 80 ppm and 500 ppm synthetic hardwater achieved a >6.95 log reduction (no survivors) after a 1.0 minuteexposure time at 50° C., while achieving an average log reduction of2.34 after 30 seconds. At 40° C., Example A achieved an average logreduction of 2.16 when diluted in both 80 ppm and 500 ppm synthetic hardwater at all exposure times. To achieve total kill of Arthriniumsacchari ascospores, Example A should be used at a concentration of 2.2%at 50° C., utilizing an exposure time of 1.0 minute.

The foregoing discussion and Examples are illustrative of the invention.However, since many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides wholly in the claims hereinafter appended.

We claim:
 1. A cold aseptic bottling method for beverage containers thatobtains a significantly reduced population of fungal microorganisms,said fungal organisms posing a substantial contamination risks in coldaseptic bottling, said method resulting in a sanitized beveragecontainer, the method comprising contacting a beverage container with aperoxyacid antimicrobial composition comprising a mixed peroxyacidsanitizer comprising at least 10 ppm of peracetic acid and at least 1ppm of a C₆-C₁₈ peroxyacid for sufficient period of time to reduce thefungal microorganism contaminating population comprising a fungalmicroorganism selected from the group consisting of a fungus of thegenus Arthrinium, a fungus of the genus Chaetomium and mixtures thereof,rinsing the container and filling the container with a carbonated ornon-carbonated beverage followed by a sealing step.
 2. The method ofclaim 1 wherein the population is reduced by more than 5 log₁₀, and saidmixed peroxyacid sanitizer is obtained by diluting a antimicrobialconcentrate composition comprising: (a) an effective biocidal amount ofperacetic acid; and (b) an effective biocidal amount of an aliphaticC₆-C₁₈ peroxyacid; wherein the concentrate composition has aproportional weight ratio of about 20 to 1 parts of (a) per part of (b)and is capable of being diluted with a major proportion of water to formsaid mixed peroxyacid sanitizer having a pH in the range of about 2 to8.
 3. The method of claim 1, wherein the peracetic acid is present inabout 0.01 to 25 wt-%.
 4. The method of claim 1 wherein said C₆-C₁₈peroxyacid comprises peroxyoctanoic acid, peroxydecanoic acid,monoperoxy- or diperoxyadipic acid, monoperoxy- or diperoxysebacic acid,or mixtures thereof.
 5. The method of claim 1 wherein the weight ratioof said peracetic acid to said C₆-C₁₈ peroxyacid is about 15:1 to 3:1.6. The method of claim 1 wherein said mixed peroxyacid sanitizer isobtained by diluting a peroxyacid antimicrobial concentrate compositioncomprising an effective biocidal amount of peracetic acid, an effectivebiocidal amount of a C₆-C₁₈ peroxyacid, and an effective amount of ahydrotrope coupling agent that is capable of solubilizing said C₆-C₁₈peroxyacid when the concentrate composition is diluted with water. 7.The method of claim 1 wherein said mixed peroxyacid sanitizer isobtained by diluting a peroxyacid antimicrobial concentrate compositioncomprising an effective biocidal amount of peracetic acid, an effectivebiocidal amount of a C₆-C₁₈ peroxyacid, and about 1 to 50 wt-% ofhydrogen peroxide.
 8. The method of claim 1 wherein the mixed peroxyacidsanitizer has a pH in the range of about 2 to
 8. 9. The method of claim8 wherein said mixed peroxyacid sanitizer is obtained by diluting aperoxyacid antimicrobial concentrate composition comprising 0.01 to 25wt-% of peracetic acid and an effective biocidal amount of a C₆-C₁₈peroxyacid.
 10. The method of claim 8 wherein said C₆-C₁₈ peroxyacidcomprises peroxyoctanoic acid, peroxydecanoic acid, monoperoxy- ordiperoxyadipic acid, monoperoxy- or diperoxysebacic acid, or mixturesthereof.
 11. The method of claim 8 wherein the weight ratio of saidperacetic acid to said C₆-C₁₈ peroxyacid is about 15:1 to 3:1.
 12. Themethod of claim 8 wherein said mixed peroxyacid sanitizer is obtained bydiluting a peroxyacid antimicrobial concentrate composition comprisingan effective biocidal amount of peracetic acid, an effective biocidalamount of a C₆-C₁₈ peroxyacid, and an effective amount of a hydrotropecoupling agent that is capable of solubilizing said C₆-C₁₈ peroxyacidwhen the concentrate composition is diluted with water.
 13. The methodof claim 8 mixed peroxyacid sanitizer is obtained by diluting aperoxyacid antimicrobial concentrate composition comprising an effectivebiocidal amount of peracetic acid, an effective biocidal amount of aC₆-C₁₈ peroxyacid, and about 1 to 50 wt-% of hydrogen peroxide.
 14. Themethod of claim 1 wherein the fungus is of the genus Chaetomium.
 15. Themethod of claim 1 wherein the fungus is of the genus Arthrinium.
 16. Themethod of claim 1 wherein the beverage is carbonated.
 17. The method ofclaim 1 wherein the beverage is a noncarbonated fruit drink or a tea.